This invention relates to a method for determining ion channel activity of substances such as peptides, polypeptides and proteins and to a method for screening potential therapeutic substances for their ability to modulate ion channel function.
Biological cells are encapsulated in a membrane made of a double layer of lipids separating the intracellular contents from the outside. The lipid bilayer xe2x80x9csandwichxe2x80x9d has a hydrophobic interior that prevents movement of charged particles such as ions across it. However, there are protein macromolecules that penetrate the membrane and act as portholes to allow ions to pass between the inside and outside of a cell. These structures that allow rapid movements of ions (many millions per second) across a cell membrane, with no need for an immediate energy input, are called xe2x80x9cion channelsxe2x80x9d. The forces that influence the movement of ions through a channel are electrical and chemical. The electrical force is the electrical potential across the membrane, the chemical force is the difference in concentration of an ion on the two sides of the membrane: the combination of the two is the electrochemical gradient for an ion. If the electrochemical gradient for an ion is not zero, ions will flow through a channel when it opens (as long as the channel lets them through).
There are many varieties of ion channels that differ in their selectivity, methods of gating, conductance and kinetic properties. Channels can be selective for sodium ions, or for potassium ions, or for calcium ions, or for chloride ions, or for protons etc and are classified according to the ions that pass through them most freely. For example, sodium channels are more permeable to sodium than to any other cations or anions. Channels are also classified according to the way in which they are turned on or gated. For example, voltage-activated channels open or close in response to changes in membrane potential. Ligand-gated channels are turned on when ligands such as neurotransmitters or hormones bind to their surface. Proteins to which ligands bind are commonly called receptors and many receptors are part of the same macromolecule that forms the ion channel. However, some channels are indirectly linked to receptors by second messenger systems and the channel is then separate from the receptor. Channels can also have very different conductances. Conductance, the reciprocal of resistance, is a measure of the ease with which ions pass through a channel and is given by the ratio of the current to the driving force. The conductance of different channels can range from picosiemens to hundreds of picosiemens (corresponding to resistances of 109 to 1012 ohms). Finally, channels can have very different xe2x80x9cduty cyclesxe2x80x9d. Some are open most of the time while others open infrequently. Some flicker rapidly between open and closed states while others do not. Changes in the environment of channels (e.g the presence of drugs) can change these characteristics. Indeed it is becoming clear that many drugs exert their effects on cells and organs by binding to surface receptors and influencing channel behaviour.
The function of all cells in an animal or other organism depends on the ion channels formed by membrane proteins which provide a pathway for movement of ions between compartments in a cell and between the interior and exterior of cells. These movements of ions are essential for normal cell function, and all biological cells (including bacteria and even enveloped viruses such as the influenza and HIV viruses) contain ion channels. Ion channels are fundamental to cellular functions such as transmission of signals in nervous systems, cell division, production of antibodies by lymphocytes, replication of virus particles within cells and secretion of fluid and electrolytes.
A wide variety of diseases such as cystic fibrosis, muscular dystrophies, stroke, epilepsy and cardiac arrhythmias are related to disorders of ion channel function. In addition, it has recently been discovered that some viruses have proteins that form ion channels that are needed in the normal life-cycle of the virus. For example, there is now good evidence that a protein (M2) in influenza A virus forms an ion channel that is necessary for virus replication, and drugs such as amantadine that block this channel inhibit replication of the influenza A virus. Amantadine (1-aminoadamantane hydrochloride) and its analogue rimantidine have been found empirically to be effective in the prophylaxis and treatment of influenza caused by the influenza A virus. These drugs, at the therapeutic concentrations, inhibit replication of the influenza A virus both in vitro and in vivo. However, they can become ineffective because of the development of resistant strains of the virus and this reduces their value as therapeutic agents.
Other drugs which work by modulating ion channel function include calcium channel blockers which are used as anti-anginal and antihypertensive agents, barbiturates which cause sleep and inhibit epileptic seizures by increasing movements of chloride ions across receptors activated by gamma-amininobutyric acid (GABA), and benzodiazepines which relieve anxiety and produce anaesthesia by increasing GABA receptor activity.
In the past, the discovery of drugs which block ion channels has been largely serendipitous. Drugs that have been discovered in this way include general anaesthetics such as ether and halothane, the barbiturates and benzodiazepines. Thus, ether was originally used like alcohol at parties, and the reversible anaesthetic effect of halothane was discovered during leakage of refrigerant from a compressor. Similarly, the discovery of the antiarryhthmic action of quinidine followed use of quinine as an antimalarial drug.
Realisation that ion channels could prove to be an important site of drug action has lead to a search for effective ways of screening the activity of potential therapeutic substances that affect ion channel activity. Although electrophysiological techniques can be used to detect current flow when ions move across channels, the methods are too tedious and time-consuming for routine screening of ion channel activity.
Vpu is a small phosphorylated integral membrane protein encoded by the HIV-1 genome which associates with the Golgi and endoplasmic reticulum membranes in infected cells, but has not been detected in the plasma membrane nor in the viral envelope. The protein is 80-82 amino acids long depending on the viral isolate, with an N-terminal transmembrane anchor and a hydrophilic cytoplasmic C-terminal domain. The C-terminal domain contains a 12 amino-acid sequence that is conserved in all isolates and contains two serine residues that are phosphorylated. Using standard techniques associated with reconstitution of the purified HIV-1 Vpu protein in planar lipid bilayers, it has been shown that the Vpu protein forms cation selective ion channels in phospholipid bilayers (8). Further work is now directed to finding drugs that block these channels, and testing them as potential anti-HIV-1 therapeutic agents. While screening for such drugs is possible using the above mentioned planar lipid bilayer method, this method has the disadvantage of requiring large quantities of highly purified Vpu protein and is limited in that only one compound can be tested per bilayer, making it a relatively slow and inefficient screening assay.
Because of these disadvantages, there is a need for an ion channel assay system that can be used both to detect the ion channel activity of biologically important peptides and proteins, and to screen the effectiveness of potential therapeutic substances that might interact with ion channels and modulate ion channel function.
Some organisms such as bacteria accumulate amino acids and other substances by using the energy of a cation concentration gradient. If a substance such as a peptide, polypeptide or protein that forms a channel is inserted in the cell membrane and dissipates the gradient, the organism can no longer accumulate essential substances and growth is inhibited. This growth inhibition can be detected directly. Thus, the activity of potential therapeutic substances that might influence the function of a channel can be quickly screened by examining their effects on growth of an organism containing the channel-forming peptide, polypeptide or protein.
In one aspect, the present invention provides a method for determining ion channel activity of a substance which is a peptide, polypeptide, protein or the like, which comprises the steps of:
(i) expressing said substance as a heterologous protein in a host cell; and
(ii) determining changes in permeability of the plasma membrane of said host cell induced by expression of said heterologous protein.
Preferably, the determination of changes in the permeability of the plasma membrane of the host cell is carried out by detecting changes in the permeability of the plasma membrane to small metabolite molecules, for example proline or adenine.
In accordance with this aspect of the invention, if the test substance is expressed as a heterologous protein having ion channel activity, expression of the heterologous protein in the plasma membrane of the host cell will alter the ability of the cell to maintain concentration gradients of small metabolite molecules such as proline or adenine whose transport into the cell is energised by the ions which are permeable to the expressed channel. As a result, a net movement or leakage of the metabolite molecules out of the cell will occur, and such leakage of the metabolite molecules can then be detected by a suitable method. Preferred methods for detecting leakage of the metabolite molecules from the cell are described below.
In a further aspect, the present invention provides a screening method for determining ion channel modulating activity of a test substance, which comprises the steps of:
(i) expressing a substance having ion channel activity as a heterologous protein in a host cell;
(ii) contacting said host cell with the test substance; and
(iii) determining changes in ion channel activity of said heterologous protein induced by the test substance.
Preferably, in this aspect of the invention, changes in ion channel activity of the heterologous protein induced by the test substance are determined by detecting the effect of the test substance on changes in permeability of the plasma membrane of the host cell induced by expression of the heterologous protein in the cell; in particular, by detecting the effect of the test substance on changes in the permeability of the plasma membrane of the host cell to small metabolite molecules such as proline, adenine or the like.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word xe2x80x9ccomprisexe2x80x9d, or variations such as xe2x80x9ccomprisesxe2x80x9d or xe2x80x9ccomprisingxe2x80x9d, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
In a preferred embodiment, the assay method of the present invention measures the alteration of the permeability to small metabolite molecules (proline or adenine, for example) of the plasma membrane of living host cells (E. coli, for example) induced by the expression of heterologous cation (sodium, for example) channel proteins (Vpu, for example) in the host cells. Although the following detailed description is directed specifically to Vpu ion channels, it will be understood that the concept of the assay is generally applicable to any ion channel protein that can be actively expressed in a host cell such as E. coli. 
The plasma membrane of a cell generally contains proteins whose function is the uptake of metabolite molecules into the cell. In a subset of these proteins, the energy to drive the uptake reaction is derived from transmembrane concentration gradients of various ions (eg Na+, H+) such that the movement across the membrane and into the cell of the metabolite to be taken up is tightly coupled to the movement across the membrane of an ion moving down its concentration gradient. If a heterologous channel forming protein is present in the membrane of the cells and causes the dissipation of the concentration gradient of the ion driving the uptake of a metabolite, then a net movement of the metabolite out of the cell should occurxe2x80x94particularly in the case where the metabolite can be derived biosynthetically by the cell. Leakage of the metabolite from cells expressing the ion channel can be detected, for example : (i) by either the ability of the leaked metabolite to support the growth of a second type of cell that has an auxotrophic requirement of the leaked metabolite; or (ii) in the case where biosynthesis of the metabolite is rate limiting to growth, by the failure of cells expressing the heterologous channel forming protein to thrive in the absence of externally supplied metabolite.
As a specific example of the first detection method, the E. coli proline transporter is driven by the co-transport into the cell of sodium ions with proline. Cross-feeding between a strain of E. coli expressing the HIV-1 Vpu proteinxe2x80x94which consequently leaks proline due to dissipation of the sodium gradientxe2x80x94and a second strain of E. coli that cannot synthesise proline but instead must take it up from the external medium has been demonstrated. Such experiments are performed in proline deficient medium so that the only possible source of proline is via biosynthesis in the Vpu-expressing strain.
As a specific example of the second detection method, the expression of Vpu in E. coli strain XL-1 Blue at 37xc2x0 C. makes cell growth dependent on externally supplied adenine. The same strain in the absence of Vpu expression grows well when adenine is absent from the growth media.
The in vivo assay of ion channel function described above also has the advantages of speed and efficiency over the planar lipid bilayer assay (8) as a method for screening potential therapeutic substances that might block, inhibit or otherwise modulate the ion channel function as many (hundreds) such substances can be screened in a single experiment. Thus the present invention also provides a method for rapidly screening compounds for their ability to block, inhibit or otherwise modulate the function of ion channel proteins expressed in living cells.
As previously described, the assay method relies on expression of the ion channel forming proteins in the plasma membrane of the cells, altering the ability of the cell to maintain concentration gradients of the metabolites whose transport into the cells is energised by the ions which are permeable to the expressed channel. Leakage of the metabolite from the cell is preferably detected by one of two methods:
(i) cross-feeding of a second strain of cells which are auxotrophic for the leaked metabolite; or
(ii) failure to thrive of the cells expressing the ion channel in the absence of the leaking metabolite supplied in the external medium.
Preferably, the expression system involves the expression of ion channel proteins in E. coli from their corresponding genes (preferably cDNA segments) cloned into E. coli plasmid expression vectors. Such vector construction and expression in E. coli uses the standard methods associated with E. coli genetics and molecular biology, described by way of example, by Sambrook et al.(9).
One preferred embodiment of the method of the present invention arises from the observation of cross-feeding between two cell linesxe2x80x94preferably bacterial cellsxe2x80x94induced in response to ion channel activity of the expressed foreign gene(s). In the specific case where E. coli cells are being used and a sodium channel is being expressed (for example as detailed further below), the leakage of proline (a metabolite whose transport into cells is energised by the sodium gradient) from the channel-expressing cells can be detected by cross-feeding of a second strain of E. coli that is auxotrophic for proline (i.e. unable to synthesise proline). Control experiments to establish that the expressed channel is not inducing a non-specific leak of all small molecules through the cell membrane would be set up identically to detect methionine leakage. The E. coli methionine transporter is energised by ATP hydrolysis and therefore the absence of a sodium gradient should not induce leakage of methionine out of the cells.
As described above, the present invention also extends to a method for screening potential therapeutic substances that may act as ion channel inhibitors. This screening method is a simple extension of the assay method described above, which in one preferred embodiment involves setting up the cross-feeding assay in the same way as previously described, with the addition of the various substances to be tested to the cells expressing the ion channel protein. Substances which block or inhibit the ion channel activity would prevent dissipation of the permanent ion gradient, and would thereby not induce leakage of metabolites. Control experiments could be performed simultaneously to ensure the substances being tested do not affect the normal growth of E. coli. If such substances are found, they would be excluded from screening by the cross-feeding assay.
Further features of the present invention are more fully described in the following Example(s). It is to be understood, however, that this detailed description is included solely for the purposes of exemplifying the present invention, and should not be understood in any way as a restriction on the broad description of the invention as set out above.